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Biosynthesis, purification, and biochemical properties of an alkaline-thermostable keratinase derived from Bacillus cereus J6

Rongxian Zhang

Rongxian Zhang

  • School of Ecological Engineering, Guizhou University of Engineering ScienceBijie, China
  • Key Laboratory of Biological Resources Development and Ecological Restoration in Guizhou ProvinceBijie, China
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Zhang, Rongxian
, 
Chengguo Fu

Chengguo Fu

  • Key Laboratory of Biological Resources Development and Ecological Restoration in Guizhou ProvinceBijie, China
  • School of Mechanical Engineering, Guizhou University of Engineering ScienceBijie, China
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Fu, Chengguo
, 
Yipeng Feng

Yipeng Feng

Mechatronic Engineering and Intelligent Manufacturing College, Henan Open UniversityZhengzhou, China
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Feng, Yipeng
, 
Bian Hu

Bian Hu

  • School of Ecological Engineering, Guizhou University of Engineering ScienceBijie, China
  • Key Laboratory of Biological Resources Development and Ecological Restoration in Guizhou ProvinceBijie, China
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Hu, Bian
, 
Yizhong Zhang

Yizhong Zhang

  • School of Ecological Engineering, Guizhou University of Engineering ScienceBijie, China
  • Key Laboratory of Biological Resources Development and Ecological Restoration in Guizhou ProvinceBijie, China
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Zhang, Yizhong
, 
Wei Zhang

Wei Zhang

  • School of Ecological Engineering, Guizhou University of Engineering ScienceBijie, China
  • Key Laboratory of Biological Resources Development and Ecological Restoration in Guizhou ProvinceBijie, China
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Zhang, Wei
 e 
Qunying Xiao

Qunying Xiao

  • School of Ecological Engineering, Guizhou University of Engineering ScienceBijie, China
  • Key Laboratory of Biological Resources Development and Ecological Restoration in Guizhou ProvinceBijie, China
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Xiao, Qunying
  
17 mar 2025
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Pubblicato online: 17 mar 2025
Ricevuto: 30 ott 2024
Accettato: 10 feb 2025
DOI: https://doi.org/10.2478/amns-2025-0831
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© 2025 Rongxian Zhang et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

Phylogenetic tree of Strain J6 constructed via Neighbor-joining method
Phylogenetic tree of Strain J6 constructed via Neighbor-joining method

Figure 2.

Keratinase production by B. cereus J6. A. Enzyme production profile; B, Feather; C, Nitrogen sources; D, Urea; E, Carbon sources; F, Lactose; G, Mineral salts; F, MgSO4.
Keratinase production by B. cereus J6. A. Enzyme production profile; B, Feather; C, Nitrogen sources; D, Urea; E, Carbon sources; F, Lactose; G, Mineral salts; F, MgSO4.

Figure 3.

Impact of cultural conditions on B. cereus J6 producing keratinase
Impact of cultural conditions on B. cereus J6 producing keratinase

Figure 4.

SDS-PAGE analysis of the purification of J6 keratinase. Lane M, protein marker; Lane 1, the crude B. cereus J6 keratinase; Lane 2, ASP; Lane 3, the target enzyme.
SDS-PAGE analysis of the purification of J6 keratinase. Lane M, protein marker; Lane 1, the crude B. cereus J6 keratinase; Lane 2, ASP; Lane 3, the target enzyme.

Figure 5.

Impact of temperature and pH
Impact of temperature and pH

Figure 6.

The enzymatic activity of the keratinase towards various substrates
The enzymatic activity of the keratinase towards various substrates

Figure 7.

The kinetics of the purified keratinase
The kinetics of the purified keratinase

Effects of chemicals on J6 keratinase

Chemicals Concentration Relative activity (%)
Na+ 5 mmol/L 102.59±2.78
K+ 5 mmol/L 99.03±2.93
Mn2+ 5 mmol/L 106.73±2.25
Ca2+ 5 mmol/L 122.56±2.19
Mg2+ 5 mmol/L 131.81±5.14
Zn2+ 5 mmol/L 118.52±3.52
Cu2+ 5 mmol/L 60.87±4.53
Al3+ 5 mmol/L 62.67±6.21
Fe2+ 5 mmol/L 52.89±5.35
Fe3+ 5 mmol/L 31.04±3.87
Tween 20 1% 115.67±3.14
Tween 60 1% 175.27±1.31
Tween 80 1% 150.16±2.31
Triton X-100 1% 106.06±0.52
SDS 1% 84.30±2.56
DTT 1 mmol/L 155.96±2.73
EDTA 1 mmol/L 5.05±2.21
PMSF 1 mmol/L Not detected

Results of orthogonal experiment

Number A B C D Keratinase yield (U/mL)
1 1 1 1 1 375.55±3.12
2 1 2 2 2 179.8±5.93
3 1 3 3 3 359.9±6.49
4 2 1 2 3 401.4±9.84
5 2 2 3 1 342.55±3.16
6 2 3 1 2 208.9±9.53
7 3 1 3 2 186.9±42
8 3 2 1 3 307.25±4.78
9 3 3 2 1 294.9±2.65
K1 305.08 321.28 297.23 337.67
K2 317.62 276.53 292.03 192.86
K3 263.02 288.9 296.45 356.18
R 54.6 32.38 5.2 163.32
Order of priority: D > A > B > C
Optimal Combination: A2B1C1D3

Orthogonal experimental design of four factors and three levels

Level Factor
A feather (g/L) B Lactose (g/L) C Urea (g/L) D MgSO4 (g/L)
1 7.5 2.5 7.5 0.9
2 10 5 10 1
3 12.5 7.5 12.5 1.1

Purification of the keratinase produced by B_ cereus J6

Procedures Protein quantity (mg) activity (U) Specific activity (U/mg) Purification fold Recovery rate (%)
Supernatant 53.25 24032 451.31 1.00 100
ASP 15.41 14256 925.19 2.05 59.32
DEAE-650 M chromatography 0.65 2739 4215.24 9.34 11.36
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Scienze biologiche, Scienze della vita, altro, Matematica, Matematica applicata, Matematica generale, Fisica, Fisica, altro
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